GSM7977840: in vitro, tRFs released from synthesized tRNA, replicate 3; Homo sapiens; OTHER
Synthetic tRNAs were incubated with RNase 2 and the digestion products from five selected tRNAs were purified separately by equal volume of acid-phenol: chloroform and pelleted by ethanol precipitation. The pellets were later dissolved in DEPC-treated water and mixed altogether. The mixed samples were firstly treated with CIP to remove the phosphates on both terminals and then we added T4 PNK, supplemented with ATP, to ensure that all tRFs started with a 5'monophosphate and ended with 3' hydroxyl. Following purification by the acid-phenol procedure (chloroform extraction and ethanol precipitation), 100 ng of the mixture were ligated with adaptors, reverse transcription and amplification was performed according to the manufacturer's protocol of NEBNext® Multiplex Small RNA Library Prep Set for Illumina®.